Naturally occurring chloramphenicol resistance in bacteria is normally due to the presence of the antibiotic inactivating enzyme chloramphenicol acetyltransferase (CAT) which catalyzes the acetyl-S-CoA-dependent acetylation of chloramphenicol at the 3-hydroxyl group. The product 3-acetoxy chloramphenicol does not bind to bacterial ribosomes and is not an inhibitor of peptidyltransferase. The synthesis of CAT is constitutive in E. coli and other Gram-negative bacteria which harbor plasmids bearing the structural gene for the enzyme, whereas Gram-positive bacteria such as staphylococci and streptococci synthesize CAT only in the presence of chloramphenicol and related compounds, especially those with the same stereochemistry of the parent compound and which lack antibiotic activity and a site of acetylation (3-deoxychloramphenicol). Studies of the primary structures of CAT variants suggest a marked degree of heterogeneity but conservation of amino acid sequence at and near the putative active site. All CAT variants are tetramers composed in each case of identical polypeptide subunits consisting of approximately 220 amino acids. The catalytic mechanism does not appear to involve an acyl-enzyme intermediate although one or more cysteine residues are protected from thiol reeagents by substrates. A highly reactive histidine residue has been implicated in the catalytic mechanism.

Chloramphenicol Acetyltransferase: Enzymology and Molecular Biology

The enzymatic acetylation of chloramphenicol by extracts of R factor-resistant Escherichia coli.

Approximately 1 in 4 therapeutic agents are marketed as racemic mixtures, the individual enantiomers of which frequently differ in both their pharmacodynamic and pharmacokinetic profiles. The use of racemates has become the subject of considerable discussion in recent years, and an area of concern for both the pharmaceutical industry and regulatory authorities. The use of single enantiomers has a number of potential clinical advantages, including an improved therapeutic/pharmacological profile, a reduction in complex drug interactions, and simplified pharmacokinetics. In a number of instances stereochemical considerations have contributed to an understanding of the observed pharmacological effects of a drug administered as a racemate. However, relatively little is known of the influence of patient factors (e.g. disease state, age, gender and genetics) on drug enantiomer disposition and action in man. Examples may also be cited where the use of single enantiomers, nonracemic mixtures and racemates of currently used agents may offer clinical advantages. The issues associated with drug chirality are complex and depend upon the relative merits of the individual agent. In the future it is likely that a number of existing racemates will be re-marketed as single enantiomer products with potentially improved clinical profiles and possible novel therapeutic indications.

Drug Chirality and its Clinical Significance

Despite considerable advances in our knowledge of antimetabolites within the past decade and the general orientation of efforts in this field toward chemotherapy, the most effective antimicrobial agents for the treatment of infections remain in the category of substances which are elaborated by other microorganisms, namely, the antibiotics. The uniform failure of the multitudinous synthetic antimetabolites to provide satisfactory chemotherapeutic agents must signify a serious void in our understanding of important factors which permit not only a marked in vivo suppressive effect on the microorganism but also a differential inhibition between invading microorganism and host. It would seem logical then to examine the action of such antibiotics as exhibit the desired properties for clues which might lead to an extension of current general concepts of antimetabolic action. The antibiotic, chloramphenicol, seems a most suitable subject of study. It is a relatively simple, stable compound whose chemical structure is known in detail (Rebstock et al., 1949; Controulis et al., 1949). Moreover, much information has accumulated concerning the effect of structural alterations on its antimicrobial action (Collins et al., 1952; Carrara et al., 1950, 1951). Its dramatic suppressive effect on the growth of widely divergent microorganisms suggests that it may interfere with a process of fundamental importance among microorganisms. An indication of the nature of an important metabolic process affected by chloramphenicol was found in the earlier observation that this antibiotic inhibited the formation of adaptive enzymes in E8cherichia coli (Hahn and Wisseman, 1951). This effect was interpreted to indicate a possible interference with protein synthesis. Subsequent studies in this laboratory (Wisseman et al., 1952, 1953; Hahn et al., 1952) as well as by Gale and Folkes (1953) have provided confirmatory evidence. The mechanisms of protein synthesis are poorly understood at present. Undoubtedly, interference with any one of a number of different contributory processes could result in the failure of an organism to synthesize proteins. On this account, it became evident early in the course of these studies that some general experimental approach of a systematic nature would be necessary in order to eliminate, or accept, a number of possibilities regarding the action of chloramphenicol. Through an attack of this kind a reasonable distinction might be made between a direct inhibitory effect upon protein synthesis and an action of the drug affecting this process merely as a secondary manifestation by the inhibition of a reaction far removed from the final stages of protein assembly. The approach finally accepted utilized the concept of a subdivision of cellular activities into more or less traditional categories of function, each of which could be studied independently. A skeletonized and diagramnatic representation of these categories and their posible interrelations are shown in figure 1. Thus, by examining the effect of various nutrients on drug action and the action of the drug on aimilation, oxidation, production and utilization of high energy phosphate bonds, and the synthesis of carbohydrates, proteins, and nucleic acids, it has been possible to demonstrate that while all except one of the processes tested proceed without serious alteration in the presence of the drug, protein synthesis alone is dramatically suppressed. In the framework of this kind of systematic examination, failure of the drug to affect other proceses assumes considerable importance in ascribing significance and specificity to positive findings.

Mode of action of chloramphenicol. I. Action of chloramphenicol on assimilation of ammonia and on synthesis of proteins and nucleic acids in Escherichia coli.

Determination of the antibiotic chloramphenicol in meat and seafood products by liquid chromatography-electrospray ionization tandem mass spectrometry.

Bartz Quentin R

Papers

Chloramphenicol (Chloromycetin).1 IV.1a Chemical Studies

The Introduction of Isobutyl Groups into Phenols, Cresols and Homologous Compounds

An Antibiotic Related to the Xanthomycins

Acetyl derivatives of the lactones of monobasic sugar acids